species-specific ribosomal RNA (rRNA) of mycobacteria. After the rRNA is released from the organism by sonication, the labeled DNA probe combines with the target organism's rRNA to form a stable DNA:RNA hybrid. The selection reagent allows for the differentiation of non-hybridized and hybridized probe. The labeled DNA:RNA hybrids are measured in a luminometer. Probes for the identification of M. tuberculosis complex, M. avium, M. intracellulare, M. avium complex, M. kansasii, and M. gordonae are available. This method can not be used directly on fresh clinical specimens.
2-4. Chromatography analysis
Gas-liquid chromatography and its commercially available modification (Microbial Identification System; Microbial ID, Inc., Newark, NJ.) have been used in the U.S.laboratories8). Fatty acids are extracted from mycobacteria and analyzed by gas-liquid chromatography. A strong base combined with heat and pressure cleaves fatty acids from lipids in the cell wall. These fatty acids are converted to sodium salts, which are methylated and form volatile methyl esters. The extract is analyzed on a gas chromatograph. The fatty acid profile is compared by a computer pattern recognition program with a library of known species-specific patterns. At present, the system can identify 27 species of mycobacteria with good accuracy. It takes approximately 1 hour of preparation time and 30 min of chromatography time to identify mycobacteria. HPLC method9), using analysis of mycolic acids as bromophenacyl esters, can provide distinct patterns for individual species.
2-5. Immunochromatography using anti-MPB64 monoclonal antibodies
Immunogenic protein MPB64 has only been found in unheated culture filtrates of the M. tuberculosis complex except for some vaccine strains of M. bovis BCG. Monoclonal antibodies directed to a recombinant MPB64 antigen are reacted specifically with culture filtrates of M. tuberculosis. The sensitized colloidal gold and a second monoclonal antibody are coated to nitrocellulose membrane in about 2 cm distance. Then the membrane is inserted in the cassette. One hundred microliters of a mycobacterial culture are applied to the test cassette and they are allowed to stand at room temperature for more than 10 min. A brownish precipitin line is distinctly demonstrated against all M. tuberculosis cultures, but not in the cultures with MOTT bacilli
3. Gene amplification for detection of M. tuberculossis
Several gene amplification procedures have been described for use with M. tuberculosis and include polymerase chain reaction (PCR)10), transcription-mediated amplification (TMA or MTD)11), strand displacement, amplification (SDA)12), ligase chain reaction (LCR)13) reporter phage system14), and Q-beta replicase amplification15).
3-1. polymerase chain reaction
This ingenious method uses repeated cycles of oligonucleotide-directed DNA synthesis to carry out in vitro replication of target nucleic acid sequences. PCR uses oligonucleotide primers to direct the amplification of target nucleic acid sequences via repeated rounds of denaturation, primer annealing, and primer extension. The specificity of the amplification process lies in the choice of primers. The reaction mixture for PCR (50μl) consisted of 10mM Tris-HCI (pH 8.3), 50mM NaCl, 0.01% gelatin, 2mM MgCL2, 0.2mM each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dUTP), 0.01U of uracil DNA glycosylase (UNG), 1U of Ampli Taq DNA polymerase, and 10 μl of DNA prepared from the clinical sample. After the mixture is allowed to stand for 10 min at room temperature and then is incubated at 94℃ for 10 min, PCR is carried out in a thermalcycler for 40 cycles, each cycle consists of denaturation at 94℃ for 1 min, annealing at 65℃ for 1 min and primer extension at 72℃ for 1 min. After amplification, the reaction mixture is electrophoresed in a 2%, agarose gel. The gels are stained with ethidium bromide and visualized by UV transillumination. The automated COBAS AMPLICOR system (Roche Diagnostics) is available for the detection of the M. tuberculosis complex, M. avium, and M. intracellulare in clinical specimens.
3-2. Transcription-mediated amplification (TMA)
TMA, an isothermal target-based amplification system has been-combined with a homogenious detection method to detect M. tuberculosis in clinical specimens (Gen-Probe Incorporated). Ribosomal RNA (rRNA) is amplified via TMA, in which the rRNA target sequences are copied into a transcription complex by using reverse transcriptase. After that, RNA polymerase is used to make numerous RNA transcripts of the target sequence from the transcription complex. The process then repeats autocatalytically. Detection of the amplified products is achieved by using an acridinium ester-labeled DNA probe specific for M. tuberculosis complex in a homogenious solution hybridization assay format, which is similar to that used in the Gen-Probe Accuprobe species identification system.
3-3. Strand displacement amplification (SDA)
SDA is an isothermal amplification method (Becton Dickinson). A single strancled target DNA fragment and a DNA amplification primer bind at their complementary 3'
